(A) The conservation of residues shown in Figure 2 is mapped onto the surface of the C. thermophilum LIC G domain. The surface is shown in two different orientations with each orientation showing universally conserved residues (pink) and LIC1/2-specific residues (blue) vs LIC3-specific residues (purple). The LIC is oriented either toward the N-terminus or toward the C-terminal loops (as in Figure 1C). Conserved amino acids and the corresponding residue numbers are labeled according to the C. thermophilum LIC sequence. (B) HA-tagged fragments of human LIC1 (a.a. 1–389, 389–523) were expressed in HEK-293T cells, immunoprecipitated with an anti-HA antibody, and immunoblotted for the dynein heavy chain or HA tag. The asterisk denotes a non-specific band that reacts with the anti-HA antibody. (C) HA-tagged double and triple mutants of human LIC1 were expressed, immunoprecipitated, and analyzed as in (B) with additional immunoblotting for the dynein intermediate chain. The residue numbers shown correspond to human LIC1. Homo sapiens (H.s.) LIC1 residues correspond to C. thermophilum (C.t.) LIC as follows: H.s. D110, D112, D113 = C.t. D102, E104, D105; H.s. Y101, Y103, W120 = C.t. Y93, Y95, Y113; H.s. V316A, E317A, D319A = C.t. I303, E304, D306; H.s. E317A, K332A = C.t. E304, K319; H.s. R260A, E262A, E338A = C.t. E325, E248, K246.